Our basic goal is to understand how chromatin structure influences gene regulation. Chromatin is generally repressive in nature but its structure is manipulated by cells in a regulated way to determine which genes are potentially transcriptionally active and which genes remain repressed. This regulation depends on interactions between sequence-specific transcription factors, chromatin enzymes and chromatin. Our current work is focused on the ATP-dependent chromatin remodeling complexes, which use the free energy available from ATP hydrolysis to move nucleosomes along DNA or between DNA molecules and to exchange histone variants between nucleosomes (an example is the SWI/SNF complex). Mutations in these enzymes have been linked with cancer. Most of our work involves the use of budding yeast, Saccharomyces cerevisiae, as a model organism, but we are also involved in some mouse chromatin studies. During this Financial Year, we have published three papers in excellent journals, as well as two short reviews. We have made significant progress toward our goals. We have begun to address why there are multiple nucleosome spacing enzymes in yeast (and higher organisms), we have discovered novel nucleosomal particles (proto-chromatosomes) and, in collaboration with the Hinnebusch Lab (NICHD), we have begun to tease out the various contributions of ATP-dependent remodeling enzymes, epigenetic histone modifications and histone chaperones to maintaining promoter chromatin structure. Detailed summaries of these studies are provided below. 1. The ISW1 and CHD1 ATP-Dependent Chromatin Remodelers Compete to Set Nucleosome Spacing in vivo. ATP-dependent chromatin remodelling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. Ocampo J, Chereji RV, Eriksson PR and Clark DJ (2016). Nucleic Acids Res. 44, 4625-4635. 2. Novel Nucleosomal Particles Containing Core Histones and Linker DNA but no Histone H1. Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains 147 bp of DNA wrapped 1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and 160 bp, and then converts it to a core particle, containing 147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these proto-chromatosomes are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1. Cole HA, Cui F, Ocampo J, Burke TL, Nikitina T, Nagarajavel V, Kotomura N, Zhurkin VB, Clark DJ (2016). Nucleic Acids Res. 44, 573-581. 3. Genome-Wide Cooperation By HAT Gcn5, Remodeler SWI/SNF and Chaperone Ydj1 in Promoter Nucleosome Eviction and Transcriptional Activation. Chaperones, nucleosome remodeling complexes and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these co-factors function ubiquitously, and the impact of nucleosome eviction on transcription genome-wide, are poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple co-factors to address these issues for 200 genes belonging to the Gcn4 transcriptome, of which 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 co-chaperone Ydj1, and chromatin-associated factor Yta7 are required downstream of Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC and H2AZ are dispensable, for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double and triple mutants implicated Gcn5, Snf2 and Ydj1 in H3 eviction at most, but not all Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these 3 co-factors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in co-factor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes, but that other steps in gene activation are more rate-limiting for most other yeast genes. Qiu H, Chereji R, Hu C, Cole HA, Rawal Y, Clark DJ, Hinnebusch AG (2016). Genome Res. 26, 211-225. 4. Invited Reviews: Ocampo J, Clark DJ (2015). A Positive Twist to the Centromeric Nucleosome. Cell Rep. 13, 645-646. Gerasimova NS, Pestov NA, Kulaeva OI, Clark DJ, Studitsky VM (2016). Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription. Transcription 7, 91-95.